Project Summary Animal development is remarkably robust to many kinds of perturbations. Animals are able to successfully develop into healthy adults despite the large amount of genetic variation in the population and the environmental and intrinsic fluctuations to which the embryo is exposed during development. However, development is not infinitely robust, and even small changes, e.g. a SNP in a developmental enhancer, can lead to developmental defects. My long-term research goal is to elucidate how some changes to the regulatory DNA that encodes the developmental program are tolerated, while others have dramatic phenotypic effects. This proposal describes work to identify the mechanisms by which shadow enhancers provide developmental systems the capability to withstand perturbations. Shadow enhancers are sets of seemingly redundant enhancers that control a single gene and drive the same spatio-temporal expression pattern. Developmental genes controlled by sets of shadow enhancers can better withstand perturbations than genes controlled by a single enhancer. Shadow enhancers are a pervasive feature of development and have been found in insects, mammals, and plants. A survey of ~1000 developmental genes in Drosophila found that nearly two-thirds of examined loci contained a set of shadow enhancers. Despite their importance, there is no proven mechanism for how shadow enhancers buffer perturbations to drive robust gene expression patterns. Dissecting these mechanisms of robustness will allow us to predict what types of perturbations developmental systems can successfully buffer and what types are intolerable, and to rationalize why certain steps in development may be more fragile than others. To identify the mechanisms of shadow enhancer robustness, this proposal describes a combination of quantitative experimental measurements and mathematical modeling of shadow enhancer function under normal conditions and perturbation. The results of this work will distinguish between two hypotheses that explain how shadow enhancers drive robust patterns of gene expression in the face of both the inevitable noise that arises from discrete molecular processes and external perturbations to the system. Success in these projects will also yield insight into how specific features of shadow enhancers may be tuned to buffer specific types and amplitudes of perturbations during the developmental process. Given the prevalence of shadow enhancers in controlling developmental processes like neural crest specification and thymocyte development, understanding their mechanisms of action may give insights in to why some developmental systems are more fragile than others and may guide efforts to find causative mutations for developmental defects in non-coding DNA.